Project Summary There is a need for strategies to precisely eliminate harmful bacteria from the host without widespread disruptions to the beneficial bacterial populations. Current strategies, such as antibiotic treatment often lead to broad changes to symbiont population compositions that lead to dysbiosis, i.e., an imbalance in the abundance of particular bacteria leading to compromised host health. Therefore, this unfulfilled need contributes to the continued persistence of diseases associated with dysbiosis, such as gastrointestinal disease, infectious disease, and autoimmune disorders. The long-term goal is to increase understanding of how inter-bacterial killing can be used as a tool promote healthy host-microbe associations and eliminate pathogens, leading to therapeutics with a low risk of dysbiosis. The overall objective of this proposal is to determine the mechanism by which contact-dependent killing between bacterial symbionts is regulated during host colonization. This research objective will be accomplished using the symbiosis established between the bobtail squid and bacterium Vibrio fischeri. This natural symbiosis provides a platform to study inter-bacterial killing within a simple, tractable bacterial symbiont. Bioluminescent populations of tightly packed V. fischeri cells assemble within a specialized host structure called the light organ, where the population composition can be directly visualized. Expression of bioluminescence is controlled by the quorum-sensing signaling network, which permits communication between bacterial cells within a population. Together, these aspects of the squid-vibrio symbiosis provide the unique opportunity to study inter-bacterial killing and how that regulation influences the composition of symbiotic populations. Preliminary data suggests that quorum sensing regulates killing between V. fischeri cells through the type VI secretion system (T6SS). The central hypothesis of this proposal is that the quorum-sensing pathway regulates T6SS expression in V. fischeri during symbiosis establishment. This central hypothesis will be tested via three specific aims: 1) determine how quorum sensing impacts T6SS activity; 2) determine how T6SS structural components are transcriptionally regulated; and 3) determine the impact of T6SS expression on bacterial fitness. The experiments in Aim 1 will evaluate how quorum sensing impacts T6SS activity by testing the extent to which different components of the quorum-sensing signaling cascade impact T6SS-mediated killing. The experiments proposed in Aim 2 will determine how a vital structural component of the T6SS is regulated by investigating co-regulation by a transcriptional activator and a repressor. The experiments in Aim 3 will determine the biological significance of the regulatory link between quorum sensing and the T6SS using in vivo host colonization assays. The outcomes of the proposed studies will provide molecular insight into a mechanism that controls elimination of bacteria from a host, providing a potential avenue for a therapeutic strategy to alter symbiont compositions in a desired manner.